Inactivated vaccine against feline calicivirosis

ABSTRACT

Immunogenic preparations and vaccines, in particular which are inactivated, effective against feline calicivirosis, based on an FCV virus strain 431 as deposited at the CNCM under the accession number CNCM I-2166, or one of its equivalents, in a veterinarily acceptable vehicle or excipient, preferably combined with FCV virus obtained from another FCV strain, in particular strain G1 as deposited at the CNCM under the accession number CNCM I-2167.

[0001] The present invention relates to the use of particular strains offeline caliciviruses for the production of immunogenic preparations andof vaccines, in particular inactivated or subunit vaccines, againstfeline calicivirosis. These immunogenic preparations and these vaccinesmay also be combined with immunogenic preparations or vaccines preparedon the basis of other feline pathogens, for the production ofmultivalent immunogenic preparations and vaccines.

[0002] Feline caliciviruses (FCV) were first described in 1957 (FastierL. B. Am. J. Vet. Res. 1957. 18, 382-389). Feline caliciviruses are,with the feline herpesviruses, the two principal sources of viraldiseases of the upper respiratory tract in cats. The FCV viruses affecta large number of animals, with FCV carrying rates of the order of 15 to25%, and an anti-FCV seroprevalence of 70 to 100% (Coutts et al. Vet.Rec. 1994. 135. 555-556; Ellis T. M. Australian Vet. J. 1981. 57.115-118; Harbour et al. Vet. Rec. 1991. 128. 77-80; Reubel et al. FelineDendistry 1992. 22. 1347-1360). After an initial phase of hyperthermia,these respiratory diseases are generally accompanied by buccalulcerations (palate, tongue, lips, nose), rhinitis, conjunctivitis,possibly anorexia and asthenia. The FCV viruses can also causepneumonia, enteritis, and articular pain (lameness syndrome).

[0003] The FCV virus is transmitted only horizontally, there is novertical transmission from the mother to its kitten during gestation(Johnson R. P. Res. Vet. Sci. 1984. 31. 114-119). FCV is transmitted bycontact between infected animals and healthy animals or by the airwaysduring sneezing (Wardley RC. Arch. Virol. 1976. 52. 243-249).

[0004] Feline caliciviruses are naked viruses of the Caliciviridaefamily, they possess a single-stranded positive RNA of about 7.7kilobase pairs (kbp) in size (Radfor et al. Proc. 1^(st) Int. Symp.Caliciviruses ESVV 1997. 93-99).

[0005] Like many RNA viruses, a large heterogeneity exists within theviral population of FCV. The antigenic variations, demonstrated sincethe beginning of the 70s by cross-serum neutralization experiments, makeit possible to classify the FCVs into several viral strains orquasispecies (Radford et al. Proc. 1^(st) Int. Symp. Caliciviruses ESVV1997. 93-99).

[0006] Several FCV strains have been identified and isolated, inparticular strain F9 (deposited with the American Type CultureCollection or ATCC under the accession number VR-782), strain 2280 (ATCCVR-2057), strain KCD (ATCC VR-651) and strain CFI (ATCC VR-654).

[0007] Vaccination against FCV was introduced since the end of the 70sfrom attenuated FCV strains, mainly strain F9 isolated in the USA in1958 by Bittle (Bittle et al. Am. J. Vet. Res. 1960. 21. 547-550) orstrains derived from F9 by passage in vitro or in vivo (“F9-like”).

[0008] Inactivated vaccines are also available. They mainly use strains255 and 2280, which were isolated in the USA respectively in 1970 in acat with a pneumonia (Kahn and Gillepsie. Cornell Vet. 1970. 60.669-683; Powvey et al. J. Am. Vet. Med. Assoc. 1980. 177. 347-350) andin 1983 in a cat suffering from lameness (Pedersen et al. Fel. Prac.1983. 13. 26-35; Pedersen N. C. and Hawkins K. F. Vet Microbiol. 1995.47. 141-156).

[0009] Because of antigenic drift over time, antisera produced againstvaccine strains isolated in the 60-70s, such as strains F9, 255 or 2280,neutralize only few isolates of the 90s. For example, the anti-F9 serumneutralizes 43% of the American isolates of the period 1990-1996,against 56% for the period 1980-89 and 86% for the period 1958-79, andonly 10% of the English isolates of the period 1990-96 (Lauritzen et al.Vet. Microbiol. 1997. 56. 55-63). Accordingly, attenuated andinactivated vaccines from old FCV strains at present no longer offersufficient protection against recent FCV strains.

[0010] The objective of the present invention is the detection of newFCV strains, which induce in cats antibodies having a broadcross-neutralization spectrum.

[0011] Another objective of the invention is the production ofimmunogenic preparations and of vaccines against feline calicivirosisfrom these FCV strains.

[0012] Yet another objective of the invention is the production ofmultivalent immunogenic preparations and of multivalent vaccines againstfeline calicivirosis and against at least one other feline pathogen.

[0013] The Applicant has selected four FCV strains obtained bypharyngeal swabs taken in France, the United Kingdom and the USA on catsexhibiting signs of infection by feline calicivirus. They arerespectively strain G1 (deposited at the Collection Nationale deCultures de Microorganismes (or CNCM) of the Institut Pasteur, Paris,France, under the accession number I-2167) and strain 431 (deposited atthe CNCM under the accession number I-2166), both deposited on Mar. 12,1999. The latter two strains are American and designated RMI6 and RMI9.The FCV G1 strain isolated in France does not correspond to the FCVstrain isolated in the United Kingdom in 1978 by Tohya Y. (Tohya Y. etal. Jpn. J. Sci., 1990, 52, 955-961) and also called G1.

[0014] The selection of the FCV 431, G1, RMI6 and RMI9 strains wascarried out by cross-serum neutralization tests with respect to the FCVisolates of a reference panel. This reference panel is composed of 18current isolates of FCV taken from cats exhibiting signs of infectionwith feline calicivirus and coming from three distinct geographicalregions. 7 isolates are American, these isolates are identified RMI1,RMI2, RMI3, RMI5, RMI6, RMI7 and RMI9. 7 isolates are French, they aredesignated A2, F1, G1, G3, F3031, H3-2 and H1-4. The last 4 isolates areEnglish, they are designated 431, 388b, 337 and J5.

[0015] The panel strains and the RMI6 and RMI9 strains are accessiblefrom the Applicant simply on request.

[0016] They have also been published in a review article “Archives ofVirology” (Poulet et al. Arch. Virol. February 2000. 145(2). 243-261),available online on Internet on the date of filing with the editor.

[0017] During cross-serum neutralization tests between the 18 FCVisolates of the reference panel, it was found, surprisingly, that theantiserum for isolate 431 neutralizes 14 of the 17 heterologous isolatesof the reference panel (the homologous serum neutralization titer is nottaken into account). By comparison, the antisera for the “historical”vaccine strains 255 and F9 neutralize only 2 of the 18 panel isolateseach.

[0018] Unexpectedly, the Applicant has therefore found with the FCV 431strain a dominant strain which can be used for the protection of theFelidae and in particular of cats against most FCV strains. By virtue ofthe panel of FCV strains disclosed here, it is possible for personsskilled in the art to select other dominant FCV strains. By way ofequivalence, the invention also covers through the FCV 431 strain theFCV strains which are equivalent thereto, which have antibodies withbroad cross-neutralization spectrum.

[0019] Equivalence exists when the antiserum for an FCV strainseroneutralizes at least 13 of the 18 heterologous isolates of thereference panel (that is to say including FCV 431), preferably when itseroneutralizes at least 14 of the 18 heterologous isolates of thereference panel, still more preferably when it seroneutralizes at least15 of the 18 heterologous isolates of the reference panel.

[0020] It is generally considered that an FCV strain seroneutralizesanother FCV strain when the heterologous serum neutralization titer isgreater than or equal to 1.2 log₁₀ VN₅₀ (Povey C. and Ingersoll J.,Infection and Immunity, 1975, 11, 877-885). The Applicant took thisvalue as the positivity threshold. However, the cross-serumneutralization results obtained with an FCV isolate having a homologousserum neutralization titer of less than or equal to 2 log₁₀ VN₅₀ cannotbe interpreted.

[0021] A second method for establishing the equivalence of an FCV strainwith respect to the FCV 431 strain is to use monoclonal antibodiesspecific for the FCV 431 strain and to test the candidate FCV strain byindirect immunofluorescence (IIF). The Applicant has thus succeeded inproducing several monoclonal antibodies which have proved specific forthe 431 strain. One of them was called 44. There is equivalence if thereis reactivity in immunofluorescence with monoclonal antibodies specificfor 431, for example with the monoclonal antibody 44. This monoclonalantibody and the corresponding hybridoma are available from theApplicant upon simple request and are also disclosed in the article byPoulet et al. Arch. Virol. 2000. 145. 1-19. The corresponding hybridomawas also deposited on Aug. 11, 1999 at the CNCM under the accessionnumber I-2282. It goes without saying, however, that persons skilled inthe art are perfectly capable of producing monoclonal antibodies byconventional techniques and of selecting, relative to the panel, thosewhich are specific for the 431 strain.

[0022] The first subject of the present invention is thereforeimmunogenic preparations and vaccines prepared from feline calicivirusstrain 431, which includes its equivalents as defined above, preferablyin inactivated or subunit form, in a veterinarily acceptable vehicle orexcipient, and preferably in the presence of an adjuvant. The notion ofimmunogenic preparation covers here any preparation capable, onceadministered to cats, of inducing an immune response directed againstthe feline pathogen considered. Vaccine is understood to mean apreparation capable of inducing effective protection.

[0023] The other FCV G1, RMI6 and RMI9 strains were chosen for theircomplementarity to the FCV 431 strain, namely that the combination ofthe antisera for 431 and for one of these three FCVs seroneutralize 100%of the isolates of the reference panel, that is to say that these threeFCV strains have a homologous serum neutralization titer greater than orequal to 2 log₁₀ VN₅₀ and heterologous serum neutralization titersgreater than or equal to 1.2 log₁₀ VN₅₀ with respect to the FCV isolatesof the reference panel against which the 431 antiserum does notseroneutralize or seroneutralizes weakly (value less than 1.2 log₁₀VN₅₀). The invention also covers the equivalent FCV strains having thesame complementarity with respect to the FCV 431 strain. It is alsopossible to produce and select monoclonal antibodies specific for thesestrains, in particular for G1, which makes it possible to determineequivalents on this other basis.

[0024] The second subject of the invention is therefore immunogenicpreparations and vaccines comprising, in addition to the antigens of theFCV 431 strain or one of its equivalents according to the invention,antigens of at least one other FCV strain, especially a complementarystrain, in particular chosen from the group comprising G1, RMI6, RMI9,which includes their equivalents, in a veterinarily acceptable vehicleor excipient, and optionally an adjuvant. Preferably, the antigensobtained from the other FCV strain(s) comprise inactivated virus orsubunits.

[0025] The subject of the invention is in particular the combination ofthe two FCV 431 and G1 strains for the production of immunogenicpreparations or of inactivated or subunit vaccines.

[0026] Surprisingly, the combination of the two FCV G1 and 431 strainscauses advantageously a synergistic effect. During studies on thecomplementarity of the FCV G1 and 431 strains, the immune responsesinduced by G1 alone, 431 alone or the combination of both (G1+431) werecompared. The group of animals which were immunized with the combinationof the two FCV G1 and 431 strains had the benefit of a better clinicalprotection.

[0027] The culture and propagation of the FCV viruses is preferablycarried out on feline cells, more particularly on Crandell-Reese FelineKidney or CRFK cells (accessible from the American Type CultureCollection under the number CCL-94) with a multiplicity of infection(moi) of 2 to 0.01 cell culture infectious doses 50% (CCID₅₀) per cell,preferably 0.5 CCID₅₀/cell.

[0028] After harvesting and clarifying, the FCV viruses intended toproduce an inactivated immunogenic preparation or an inactivated vaccineare inactivated by a chemical treatment (e.g. formalin or formaldehyde,β-propiolactone, ethylenimine, binary ethylenimine (BEI)) and/or a heattreatment. Preferably, the viruses according to the invention areinactivated by the action of ethylenimine formed immediately before usefrom bromoethylamine (BEA). The viral particles may be concentrated byconventional concentration techniques, in particular by ultrafiltrationand then optionally purified by conventional purification means, inparticular gel filtration techniques or selective precipitationtechniques in particular in the presence of polyethylene glycol (PEG). Apurification without previous concentration can also be done.

[0029] For the production of an immunogenic preparation or of aninactivated or subunit vaccine, the viral particles are taken up in aveterinarily acceptable vehicle or excipient, and optionallysupplemented with an adjuvant. The quantity of antigen is in particularequal to a preinactivation titer of about 10⁵ to about 10¹⁰ CCID₅₀ perdose, preferably of about 10⁸ to about 10⁹ CCID₅₀ per dose.

[0030] To supplement the immunogenic preparations and vaccines accordingto the invention with adjuvants, it is possible to use as adjuvant (1)aluminum, hydroxide, (2) a polymer of acrylic or methacrylic acid, apolymer of maleic anhydride and of alkenyl derivative, or (3) toformulate the immunogenic preparation or vaccine in the form of anoil-in-water emulsion, in particular the emulsion SPT described p 147“Vaccine Design, The Subunit and Adjuvant Approach”edited by M. Powell,M. Newman, Plenum Press 1995, and the emulsion MF59 described p 183 inthe same book.

[0031] The oil-in-water emulsion may in particular be based on lightliquid paraffin oil (European Pharmacopeia type); isoprenoid oil such assqualane, squalene; oil resulting from the oligomerization of alkenes,in particular of isobutene or of decene; esters of acids or alcoholscontaining a linear alkyl group, more particularly vegetable oils, ethyloleate, propylene glycol di(caprylate/caprate), glyceryltri(caprylate/caprate), propylene glycol dioleate; esters of branchedfatty alcohols or acids, in particular esters of isostearic acid. Theoil is used in combination with emulsifiers to form the emulsion. Theemulsifiers are preferably nonionic surfactants, in particular theesters of sorbitan, mannide, glycerol, polyglycerol, propylene glycoland of oleic, isostearic, ricinoleic or hydroxystearic acid, which areoptionally ethoxylated, the polyoxypropylene-polyoxyethylene blockcopolymers, in particular the Pluronic® copolymers, especially L121.

[0032] The polymers of acrylic or methacrylic acid are crosslinked, inparticular with polyalkenyl ethers of sugars or polyalcohols. Thesecompounds are known under the term carbomer (Pharmeuropa vol. 8, No. 2,June 1996). Persons skilled in the art can also refer to U.S. Pat. No.2,909,462 (incorporated by way of reference) describing such acrylicpolymers crosslinked with a polyhydroxylated compound having at least 3hydroxyl groups, preferably not more than 8, the hydrogen atoms of atleast three hydroxyls being replaced with unsaturated aliphatic radicalshaving at least 2 carbon atoms. The preferred radicals are thosecontaining 2 to 4 carbon atoms, e.g. vinyls, allyls and otherethylenically unsaturated groups. The unsaturated radicals maythemselves contain other substituents, such as methyl. The products soldunder the name Carbopol® (BF Goodrich, Ohio, USA) are particularlyappropriate. They are crosslinked with an allyl sucrose or withallylpentaerythritol. Among them, there may be mentioned Carbopol® 974P,934P and 971P.

[0033] Among the copolymers of maleic anhydride and of alkenylderivative, the EMA® copolymers (Monsanto) which are copolymers ofmaleic anhydride and of ethylene, which are linear or crosslinked, forexample crosslinked with divinyl ether, are preferred. Reference may bemade to J. Fields et al., Nature, 186: 778-780, Jun. 4, 1960(incorporated by way of reference). From the point of view of their,structure, the polymers of acrylic or methacrylic acid and the EMA®copolymers are preferably formed of basic units of the followingformula:

[0034] in which:

[0035] R₁ and R₂, which are identical or different, represent H or CH₃

[0036] x=0 or 1, preferably x=1

[0037] y=1 or 2, with x+y=2

[0038] For the EMA® copolymers, x=0 and y=2. For the carbomers, x=y=1.

[0039] The concentration of polymer in the final vaccine compositionwill be from 0.01% to 1.5% W/V, more particularly from 0.05 to 1% W/V,preferably from 0.1 to 0.4% W/V.

[0040] It is of course possible to also combine inactivated virus andsubunits of the same FCV strain in accordance with the invention and/orof different FCV strains in accordance with the invention.

[0041] The subject of the invention is also a multivalent vaccinecomprising one inactivated feline calicivirus valency, comprising atleast the FCV 431 strain, which includes its equivalents, and optionallyat least one other FCV strain, in particular a strain which iscomplementary within the meaning of the invention, in particular chosenfrom G1, RMI6 and RMI9, and at least one valency for another felinepathogen, in a veterinarily acceptable vehicle or excipient andpreferably with an adjuvant, in particular one of those described above.It is likewise possible to produce subunit-based multivalent vaccines.

[0042] Said feline pathogens are in particular chosen from the groupcomprising the feline rhinotrachitis virus or the feline herpesvirus(FHV), the feline leukemia virus (FeLV), feline panleukopenia virus orfeline parvovirus (FPV), the feline infectious peritonitis virus (FIPV),the feline immunodeficiency virus (FIV), the rabies virus, Chlamydia.

[0043] The FCV vaccines according to the invention may be mixedimmediately before use with the other feline valency (valencies) whichmay be in the form of attenuated live, inactivated, subunit, recombinantor polynucleotide vaccines.

[0044] The subject of the invention is therefore also a multivalentvaccination kit or box comprising, packaged separately, an FCV valencyaccording to the invention in a veterinarily acceptable vehicle orexcipient, and preferably with an adjuvant, and at least one valency ofanother feline pathogen. The FCV valency can serve as solvent foranother feline valency, in particular attenuated, recombinant orpolynucleotide valency provided in freeze-dried form.

[0045] In accordance with a feature of the invention, it is possible toproduce immunogenic preparations or subunit vaccines, by extraction ofthe capsid from the virus, with optionally inactivation before or afterthe extraction. These preparations and vaccines therefore comprise, assole active ingredient or otherwise, such a product of extractioncontaining predominantly capsid protein and optionally subfragments,optionally inactivated, produced from the strains according to theinvention, in particular strain 431, which includes its equivalents,optionally also from another FCV strain, in particular G1 orequivalents. These subunit vaccines and preparations are advantageouslysupplemented with adjuvant, for example as described supra. It is alsopossible to mix whole inactivated vaccine or preparation and subunitvaccine or preparation.

[0046] The subject of the invention is also an immunogenic preparationor a vaccine based on the G1 strain, in particular which is inactivatedor a subunit of extraction.

[0047] The subject of the invention is also a method of immunizing catsagainst diseases caused by the feline caliciviruses.

[0048] This method of immunization comprises the administration of acombined multivalent, subunit or inactivated FCV vaccine according tothe invention to cats. The administration of said vaccine may be carriedout in particular by the parenteral route, preferably by thesubcutaneous or intramuscular route.

[0049] Persons skilled in the art have the competence necessary todefine precisely the number of injections and the doses of each vaccineto be used for each vaccination protocol.

[0050] The dose volumes may be in particular between 0.2 and 2 ml,preferably of the order of 1 ml.

[0051] The invention will now be described in greater detail with theaid of the embodiments taken by way of nonlimiting examples andreferring to the single FIGURE, giving a table of the cross-serumneutralization titers.

EXAMPLES Example 1 Viral isolates and hybridomas

[0052] The feline caliciviruses (FCV) were obtained by pharyngeal swabstaken on cats exhibiting signs of infection with feline caliciviruses.These FCVs have different geographical origins.

[0053] The FCV 431, 337, J5, 388b, 220 and 393 strains were isolated inGreat Britain and provided by Professor O. Jarrett of the University ofGlasgow, UK.

[0054] The FCV A2, G1, G3, F3031, F1, H3-2 and H1-4 strains wereisolated in France by the Applicant.

[0055] The FCV RMI1, RMI2, RMI3, RMI5, RMI6, RMI7 and FMI9 strains wereisolated in the USA by the Applicant.

[0056] The pharyngeal samples were collected in 2 ml of Dulbecco'smodified Eagle's minimum medium (DMEM, Gibco BRL), supplemented with 5%fetal calf serum (Bayer Diagnostic), with antibiotics, more particularlywith 50 mg/l of gentamycin. Each isolate is frozen at −70° C. whilewaiting to be tested. The monoclonal antibody 44, obtained from thehybridoma identified 431 2 0 17 E9 T, is specific for the FCV 431strain.

Example 2 Amplification of the viral isolates

[0057] Cells of the cat kidney line (Crandell-Reese Feline Kidney orCRFK No. ATCC CCL-94, Crandell et al. In Vitro 1973. 9. 176-185) arecultured in a 96-well plate or in a 25−cm² Falcon (Falcon) with DMEMmedium supplemented with 5% fetal calf serum, containing about 100,000cells per ml. The cells are cultured at 37° C. in an atmospherecontaining 5% CO₂. After 3 days, the cell layer arrives at confluence.The culture medium is then replaced with serum-free DMEM mediumsupplemented with 50 mg/l of gentamycin and the thawed aliquote of theFCV viral isolates (Example 1) are added at the rate of a volume of 100μl of four-fold serial dilutions per well for the limiting dilutioncloning of the FCV viruses or of 1 ml per Falcon.

[0058] When the cytopathic effect (ECP) is complete (24-48 hours afterthe start of the culture), the viral suspensions are harvested andfrozen at −70° C. 3 to 4 successive passages are generally necessary forthe production of a viral batch. The viral batch is stored at −70° C.

Example 3 Production of Serum

[0059] For each FCV virus, an antiserum was produced by inoculatingkittens by the oronasal route with 10^(6.0) CCID₅₀ of the relevant FCVvirus. The specific pathogen-free (SPF) kittens were 10 to 14 weeks old.The serum of each animal was collected one month after the infection.The sera were heat-inactivated (30 minutes at 56° C.), distributed,aliquoted and stored at −20° C.

Example 4 Cross-Serum Neutralization In Vitro

[0060] Cross-serum neutralization tests were carried out between 18field isolates obtained by pharyngeal swabs performed on cats exhibitingsigns of feline calicivirosis. 7 of them have as geographical originFrance, they are the isolates identified A2, F3031, G1, G3, F1, H3-2 andH1-4. 4 have as geographical origin Great Britain, they are the isolatesidentified J5, 337, 388b and 431. Finally, 7 have as geographical originthe USA, they are the isolates identified RMI1, RMI2, RMI3, RMI5, RMI6,RMI7 and RMI9.

[0061] The serum obtained for each isolate (Example 3) was tested forits ability to neutralize the 18 isolates. The sera were three-foldserially diluted with DMEM medium in 96-well cell culture plates. 0.05ml of culture medium containing approximately 100 CCID₅₀ of the selectedviral strain was added to 0.05 ml of the dilute serum produced as inExample 2. This mixture was incubated for 2 hours at 37° C. in anincubator under an atmosphere containing 5% CO₂.

[0062] 0.15 ml of a suspension of CRFK cells containing about 100,000cells per ml was then added to each mixture. The cytopathic effect wasobserved by phase contrast microscopy after 4 days of culture at 37° C.in an atmosphere containing 5% CO₂. The neutralizing titers of eachserum were calculated according to the Kärber method. The titers aregiven in the form of the highest dilution inhibiting the cytopathiceffect for 50% of the wells. The titers are expressed in log₁₀. Theminimum titer thus found was 0.7 log₁₀ VN₅₀. Each serum was titrated atleast twice, preferably three times.

[0063]FIG. 1 gives all the neutralizing titers obtained duringcross-serum neutralizations carried out between these 18 FCV strains andthese 18 sera.

Example 5 Indirect Immunofluorescence (IIF) Tests

[0064] The IIF tests are carried out on 96-well plates containing theCRFK cells cultured in monolayers infected with the FCV viruses to betested.

[0065] 200 μl per well of a suspension of CRFK cells containing 90,000cells/ml in F15 medium (Gibco BRL, Cat # 045-1075) containing 5% fetalcalf serum are cultured in a 96-well plate. At confluence, 320 CCID50 ofFCV are inoculated in 100 μl of F15 medium. When the first CPE fociappear, the cells are then rinsed with cold PBS with no calcium ormagnesium (PBS, Sigma), and then fixed at −20° C. for 30 minutes withcold acetone containing 5% v/v of water. After drying, the infected andfixed cells are brought into contact for 30 minutes at 37° C. with 100μl per well of ascitic fluid corresponding to the anti-FCV 431monoclonal antibody 44 (hybridoma 431 2 0 17 E9 T, diluted 1/5000approximately in 50 mM TRIS-HCL buffer, pH 7.6.

[0066] After two rinses in PBS, the attachment of the antibodies isvisualized by incubation under the same conditions of a goat anti-mouseIgG antibody conjugated with fluorescein isothianate (Biosys, FITCconjugated at 2 mg/ml) and diluted 1/150 in 50 mM TRIS-HCl buffer, pH7.6. The reading is made under an optical microscope under UV light.

[0067] This monoclonal antibody was tested with respect to each of theisolates of the panel. It is attached exclusively to the CRFK cellsinfected with FCV 431.

[0068] This test may be used to determine the equivalents of the FCV 431strain. These equivalents are those to which the monoclonal antibody 44attaches.

Example 6 Synergy

[0069] 32 nonvaccinated SPF kittens about 9 weeks old are divided byrandomization into 4 groups (identified from A to D) of 8 kittens each,each group is housed in an isolated box.

[0070] After thawing the viral suspensions (Example 2) and diluting inPBS so as to obtain the desired titer, the cats are vaccinated bysubcutaneous injection of 1 ml of FCV G1 inoculum at 10^(3.3) CCID₅₀/mlfor group B, of 1 ml of FCV 431 inoculum at 10^(3.5) CCID₅₀/ml for groupC, of 0.5 ml of FCV G1 inoculum at 10^(3.3) CCID₅₀/ml and 0.5 ml of FCV431 inoculum at 10^(3.5) CCID₅₀/ml (at a different injection site) forgroup D. Group A serves as control group.

[0071] Half of each group A to D is randomly distributed into two groups1 and 2 and housed in separate boxes. The animals are challenged on the31^(st) day after vaccination (d31).

[0072] The animals in group 1 are challenged by administration of 1 mlof challenge viral strain FCV 220 having a titer of 10^(7.2) CCID₅₀/mlby the oronasal route (0.5 ml by the oral route and 0.25 ml into eachnostril).

[0073] The animals in group 2 are challenged by administration of 1 mlof challenge viral strain FCV 393 having a titer of 10^(6.8) CCID₅₀/mlby the oronasal route (0.5 ml by the oral route and 0.25 ml into eachnostril).

[0074] The virulent strains FCV 220 and 393 were chosen because they aredistant in cross-serum neutralization from the viral strains FCV G1 and431.

[0075] Any cross-contamination between the two boxes is carefullyavoided. Clinical monitoring of the animals in both groups is done bytaking the rectal temperature and clinical examinations of the animals(general state, presence of ulcers of the tongue and of the palate,presence of gingivitis, presence of rhinitis, presence ofconjunctivitis, presence of lameness, death of the animal).

[0076] The total clinical score for each animal was calculated by addingthe scores obtained for each group of clinical signs according to thefollowing scale:

[0077] rectal temperature:

[0078] 0—less than 39° C.

[0079] 1—greater than or equal to 39° C. and less than 39.5° C.

[0080] 2—greater than or equal to 39.5° C. and less than 40° C.

[0081] 3—greater than or equal to 40° C.

[0082] general state:

[0083] 0—normal behavior

[0084] 1—exhaustion

[0085] ulcers of the tongue and of the palate (some of the diameters ofall the ulcers, if there are several):

[0086] 0—absence of ulcer

[0087] 1—diameter of 1 to 5 mm

[0088] 2—diameter of 6 to 10 mm

[0089] 3—diameter greater than 10 mm

[0090] gingivitis:

[0091] 0—absence of gingivitis

[0092] 1—gingivitis

[0093] rhinitis:

[0094] 0—absence of rhinitis

[0095] 1—rhinitis with serous nasal discharge

[0096] 2—rhinitis with mucous to mucopurulent nasal discharge

[0097] conjunctivitis:

[0098] 0—absence of conjunctivitis

[0099] 1—conjunctivitis with serous discharge

[0100] 2—conjunctivitis with mucopurulent discharge

[0101] lameness:

[0102] 0—absence of lameness

[0103] 1—lameness

[0104] death:

[0105] 0—survival

[0106] 5—death.

[0107] The mean clinical scores obtained are the following:Group/challenge FCV 220 FCV 393 Control (group A) 31 30 FCV G1 (group B)5 23 FCV 431 (group C) 6 18 FCV G1 + FCV 431 (group D) 2 9

[0108] The results thus obtained show synergy between the FCV G1 and FCV431 strains by a significant difference between the mean value obtainedfor the best strains and that obtained for the combination of the twostrains (Kruskal-Wallis test).

Example 7 Production of inactivated vaccine

[0109] The CRFK cells are cultured at 37° C. in 2-liter roller flasks(850 cm²) in modified Eagle's medium (MEM, Gibco BRL) supplemented with2.5% of lactalbumin hydrolysate (Gibco BRL) and 5% fetal calf serum(Gibco BRL) 300 ml of a cellular suspension in MEM medium containingabout 100,000 cells/ml are added per roller flask. After 3 days, thecell layer becomes confluent. The cell culture medium is then replacedwith serum-free MEM medium and the FCV virus added at a multiplicity ofinfection (moi) of 0.5 CCID₅₀/cell. The viral culture is maintained at37° C. for 24 to 48 hours until a cytopathic effect is obtained for thewhole cellular lawn. The viral suspension is harvested and thenclarified on a bag filter having a porosity of 1.5 μm. The FCV virustiter at harvest is 8.5+/−0.3 log10 CCID₅₀/ml.

[0110] The virus is inactivated with ethylenimine at the concentrationof about 8 mM at 22° C. for 18 hours.

[0111] The ethylenimine is prepared immediately before use by dissolving28 g of sodium hydroxide pellets in 200 ml of distilled water and adding68.1 g of bromoethylamine (BEA) corresponding to a 1.2 M solutionapproximately (H. Bahnemann, Arch. Virol., 1975, 47, 47-56). Theinactivated viral suspension is concentrated 100-fold on anUltrasette-type ultrafiltration cartridge with a cut-off of 100 kDa(Filtron) and then frozen at −70° C.

[0112] The inactivated viral suspension after thawing is diluted 1/33 inPBS buffer (NaCl 8 g/l; KCl 0.2 g/l; KH₂PO₄ 0.2 g/l; Na₂HPO₄, 2 H₂O 1.44g/l). The vaccine is prepared in the same manner: 167 ml of aqueousphase consisting of the dilution of the inactivated virus are emulsifiedin 83 ml of an oily phase containing 7% w/v of anhydromannitol oleate,8% w/v of ethoxylated oleic acid containing 11 molecules of ethyleneoxide (EO) on average and 85% v/v of light liquid paraffin oil (EuropeanPharmacopeia type) with the aid of a Silverson turbine emulsifier at 32°C. for 2 minutes. The vaccine is then stored at 5° C.

[0113] An alternative method for preparing the vaccine consists informing into an emulsion by three passes through a model Y110high-pressure homogenizer (Microfluidics Corp.) at a pressure of 600 barand a temperature of between 30 and 40° C. the mixture 5% w/v squalane,2.5% w/v Pluronic® L121, 0.2% w/v Tween 80, 92.3% v/v of inactivatedviral suspension diluted 1/46 in PBS buffer after thawing. The vaccineis then stored at 5° C.

[0114] Another alternative method consists in preparing a solutioncontaining 0.4% w/v of Carbopol® 974P in physiological saline (NaCl 9g/l). The pH is adjusted to 7.3-7.4 with sodium hydroxide. This solutionof Carbopol is then mixed in equal parts with the suspension ofinactivated FCV virus diluted 1/25 after thawing. The vaccine is thenstored at 5° C.

[0115] The aqueous phase of the emulsions or the aqueous phase mixedwith Carbopol® consists of a dilution in PBS of the concentratedinactivated viral suspension corresponding either to the FCV 431 strainor to the FCV G1 strain or a mixture in equal parts of the FCV 431 andG1 strains.

Example 8 Control of the Immunogenicity of Inactivated FCV 431

[0116] 19 nonvaccinated SPF kittens about 9 weeks old are divided byrandomization into 2 groups (identified from A and B), the first with 12kittens and the second with 7 kittens, each group is housed in anisolated box.

[0117] The vaccine is prepared with the adjuvant composed ofanhydromannitol oleate, ethoxylated oleic acid and light liquid paraffinoil as described in Example 7.

[0118] The cats are vaccinated twice (D0 and D28) by subcutaneousinjection of 1 ml of FCV 431 inoculum at 10⁷ CCID₅₀/ml for group A.Group B serves as control group.

[0119] The animals are challenged on the 42^(nd) day after the firstvaccination (D42) by administration of 1 ml of challenge viral strainFCV 431 having a titer of 10⁶ CCID₅₀/ml by the oronasal route (0.5 ml bythe oral route and 0.25 ml into each nostril).

[0120] The level of anti-FCV 431 neutralizing antibodies and theclinical score were monitored. The total clinical score for each animalwas calculated by adding the scores obtained for each group of clinicalsigns according to the scale given in Example 6.

[0121] The results obtained are the following:

[0122] Anti-FCV 431 neutralizing antibody titers expressed as log₁₀VN₅₀/ml: Antibody on Group Antibody on D0 Antibody on D28 D42 FCV 431vaccine 0.24 1.61 2.87 (group A) Controls 0.24 0.24 0.24 (group B)

[0123] Mean clinical scores over the period D42 to D56: Group Clinicalscore FCV 431 vaccine 0.7 (group A) Controls 33.7 (group B)

[0124] These results show an excellent clinical protection against thehomologous challenge and good seroconversion.

[0125] It should be clearly understood that the invention defined by theappended claims is not limited to the specific embodiments indicated inthe description above, but encompasses the variants which do not departfrom the scope or the spirit of the present invention.

1. Immunogenic preparation or vaccine against feline calicivirosis,based on an FCV virus having the characteristic feature of beingrecognized by the monoclonal antibody 44 deposited at the CNCM under theaccession number I-2282, and comprising a veterinarily acceptablevehicle or excipient.
 2. Immunogenic preparation or vaccine againstfeline calicivirosis, based on an FCV virus having the characteristicfeature of inducing in felines, in particular cats, an antiserum capableof neutralizing at least 13, 14 or 15 of the FCV isolates of a panelconsisting of the FCV strains identified RMI1, RMI2, RMI3, RMI5, RMI6,RMI7, RMI9, A2, F1, G1, G3, F3031, H3-2, H1-4, 431, 388b, 337 and J5,and comprising a veterinarily acceptable vehicle or excipient. 3.Immunogenic preparation or vaccine according to claim 1 or 2, based onan FCV virus of strain 431 as deposited at the CNCM under the accessionnumber CNCM I-2166, in a veterinarily acceptable vehicle or excipient.4. Immunogenic preparation or vaccine according to any one of claims 1to 3, comprising, in addition, the antigen obtained from another FCVstrain.
 5. Immunogenic preparation or vaccine according to claim 4,characterized in that the other FCV strain is strain G1 as deposited atthe CNCM under the accession number CNCM I-2167.
 6. Immunogenicpreparation or vaccine according to any one of claims 1 to 5,comprising, in addition, at least one valency against at least one otherfeline pathogen.
 7. Immunogenic preparation or vaccine according toclaim 6, characterized in that said other feline pathogen is chosen fromthe group consisting of the feline herpesviruses (FHV), the felineleukemia virus (FeLV), the feline panleucopenia virus (FPV), the felineinfectious peritonitis virus (FIPV), the feline immunodeficiency virus(FIV), the rabies virus, Chlamydia.
 8. Immunogenic preparation orvaccine according to any one of claims 1 to 7, comprising, in addition,an adjuvant.
 9. Immunogenic preparation or vaccine according to claim 8,characterized in that the adjuvant is aluminum hydroxide. 10.Immunogenic preparation or vaccine according to claim 8, characterizedin that the adjuvant is a polymer of acrylic or methacrylic acid or apolymer of maleic anhydride and of alkenyl derivative, preferably acarbomer.
 11. Immunogenic preparation or vaccine according to any one ofclaims 1 to 7, characterized in that the immunogenic preparation orvaccine is in the form of an oil-in-water emulsion.
 12. Immunogenicpreparation or vaccine according to any one of claims 1 to 11,characterized in that it comprises the inactivated FCV virus. 13.Immunogenic preparation or vaccine according to any one of claims 1 to11, characterized in that it comprises subunits obtained from the FCVstrain(s), these subunits comprising extraction capsid proteins. 14.Multivalent vaccination kit or box comprising, packaged separately, animmunogenic preparation or vaccine according to any one of claims 1 to 6and 12 to 13, preferably with an adjuvant, and at least one valency ofat least one other feline pathogen.
 15. Hybridoma as deposited at theCNCM under the accession number CNCM I-2282.
 16. Monoclonal antibodycapable of being produced from the hybridoma according to claim 15.